Biophysical Regulation of Endoderm By 3-Dimensional Fibronectin Matrix

Abstract

Fibronectin (FN), a major extracellular matrix (ECM) component that assembles into a 3-dimensional (3D) network, plays a significant role in the development and maintenance of most tissues. In the embryonic stem (ES) cell niche, ECM composition, elasticity, and architecture likely contribute to the decision between self-renewal and differentiation. ES cells differentiating as multicellular embryoid bodies (EBs) exhibit a 10-fold drop in expression of Nanog, a self-renewal marker, concurrent with a 3-fold upregulation in FN production as well as the onset of differentiation markers Fgf 5 (ectoderm), brachyury (mesoderm), and GATA4 (endoderm). However, FN and GATA4 appear to be temporally and spatially correlated within the EB while FN and Nanog are inversely correlated with each other. To probe any specific FN-GATA4 interaction and its biophysical regulation, FN-coated surfaces and 3-dimensional, soft fibrillar FN matrices were used as substrates for ES cells grown in monolayer culture. ES cells on FN-coated surfaces displayed a well spread morphology but did not significantly increase their FN production or GATA4 expression. In contrast, ES cells grown on fibrillar matrices were less spread, displayed a 4-fold upregulation of FN production similar to that of EBs, and expressed GATA4 via immunofluorescent detection. However, when crosslinked to increase 3D FN matrix elasticity from 350 Pa to 4500 Pa, FN expression dropped 2-fold and GATA4 staining was significantly reduced. Though the specific molecular mechanisms require elucidation, these findings suggest important temporal, spatial, and mechanical roles for FN matrix in regulation of ES cell development.